This invention relates to Streptococcus pneumoniae and in particular this invention relates to the identification of an S. pneumoniae protein that is capable of degrading human complement protein. C3.
This application claims the benefit of a provisional application (Ser. No. 60/044,316) filing on Apr. 24, 1997 entitled xe2x80x9cHuman complement C3-degrading proteinase from Streptococcus pneumoniae.xe2x80x9d
Respiratory infection with the bacterium Streptococcus pneumoniae (S. pneumoniae) leads to an estimated 500,000 cases of pneumonia and 47,000 deaths annually. Those persons at highest risk of bacteremic pneumococcal infection are infants under two years of age and the elderly. In these populations. S. pneumoniae is the leading cause of bacterial pneumonia and meningitis. Moreover, S. pneumoniae is the major bacterial cause of ear infections in children of all ages. Both children and the elderly share defects in the synthesis of protective antibodies to pneumococcal capsular polysaccharide after either bacterial colonization, local or systemic infection, or vaccination with purified polysaccharides. S. pneumoniae is the leading cause of invasive bacterial respiratory disease in both adults and children with HIV infection and produces hematogenous infection in these patients (Connor et al. Current Topics in AIDS 1987;1:185-209 and Janoff et al. Ann. Intern. Med 1992;117(4):314-324).
Individuals who demonstrate the greatest risk for severe infection are not able to make antibodies to the current capsular polysaccharide vaccines. As a result, there are now four conjugate vaccines in clinical trial. Conjugate vaccines consist of pneumococcal capsular polysaccharides coupled to protein carriers or adjuvants in an attempt to boost the antibody response. However, there are other potential problems with conjugate vaccines currently in clinical trials. For example, pneumococcal serotypes that are most prevalent in the United States are different from the serotypes that are most common in places such as Israel. Western Europe, or Scandinavia. Therefore, vaccines that may be useful in one geographic locale may not be useful in another. The potential need to modify currently available capsular polysaccharide vaccines or to develop protein conjugates for capsular vaccines to suit geographic serotype variability entails prohibitive financial and technical complications. Thus, the search for immunogenic, surface-exposed proteins that are conserved worldwide among a variety of virulent serotypes is of prime importance to the prevention of pneumococcal infection and to the formulation of broadly protective pneumococcal vaccines. Moreover, the emergence of penicillin and cephalosporin-resistant pneumococci on a worldwide basis makes the need for effective vaccines even more exigent (Baquero et al. J. Antimicrob. Chemother. 1991;28S;31-8).
Several pneumococcal proteins have been proposed for conjugation to pneumococcal capsular polysaccharide or as single immunogens to stimulate immunity against S. pneumoniae. Surface proteins that are reported to be involved in adhesion of S. pneumoniae to epithelial cells of the respiratory tract include PsaA, PspC/CBP112, and IgA1 proteinase (Sampson et al. Infect. Immun. 1994;62:319-324, McDaniel et al., Microb. Pathogen. 1992; 13:261-9, and Wani, et al. Infect. Immun. 1996; 64;3967-3974). Antibodies to these adhesins could inhibit binding of pneumococci to respiratory epithelial cells and thereby reduce colonization. Other cytosolic pneumococcal proteins such as pneumolysin, autolysin, neuraminidase, or hyaluronidase are proposed as vaccine antigens because antibodies could potentially block the toxic effects of these proteins in patients infected with S. pneumoniae. However, these proteins are typically not located on the surface of S. pneumoniae, rather they are secreted or released from the bacterium as the cells lyse and die (Lee et al. Vaccine 1994; 12:875-8 and Berry et al. Infect. Immun. 1994; 62:1101-1108). While use of these cytosolic proteins as immunogens might ameliorate late consequences of S. pneumoniae infection, antibodies to these proteins would neither promote pneumococcal death nor prevent initial or subsequent pneumococcal colonization.
A prototypic surface protein that is being tested as a pneumococcal vaccine is the pneumococcal surface protein A (PspA). PspA is a heterogeneous protein of about 70-140 kDa. The PspA structure includes an alpha helix at the amino terminus, followed by a proline-rich sequence, and terminates in a series of 11 choline-binding repeats at the carboxy-terminus. Although much information regarding its structure is available, PspA is not structurally conserved among a variety of pneumococcal serotypes, and its function is entirely unknown (Yother et al. J. Bacteriol. 1992:174:601-9 and Yother J. Bacteriol. 1994;176:2976-2985). Studies have confirmed the immunogenicity of PspA in animals (McDaniel et al. Microb. Pathogen. 1994; 17;323-337). Despite the immunogenicity of PspA, the heterogeneity of PspA, its existence in four structural groups (or clades), and its uncharacterized function complicate its ability to be used as a vaccine antigen.
In patients who cannot make protective antibodies to the type-specific polysaccharide capsule, the third component of complement, C3, and the associated proteins of the alternative complement pathway constitute the first line of host defense against S. pneumoniae infection. Because complement proteins cannot penetrate the rigid cell wall of S. pneumoniae, deposition of opsonic C3b on the pneumococcal surface is the principal mediator of pneumococcal clearance. Interactions of pneumococci with plasma C3 are known to occur during pneumococcal bacteremia, when the covalent binding of C3b, the opsonically active fragment of C3, initiates phagocytic recognition and ingestion (Johnston et al. J. Exp. Med 1969:129:1275-1290, Jasin H E, J. Immunol. 1972; 109:26-31 and Hostetter et al. J. Infect. Dis. 1984; 150:653-61). C3b deposits on the pneumococcal capsule, as well as on the cell wall. This method for controlling S. pneumoniae infection is fairly inefficient. Methods for augmenting S. pneumoniae opsonization could improve the disease course induced by this organism. There currently exists a strong need for methods and therapies to limit S. pneumoniae infection.
This invention relates to the identification and use of a family of human complement C3-degrading proteinases expressed by S. pneumoniae. The protein has a molecular weight of about 24 kD to about 34 kD as determined on a 10% SDS polyacrylamide gel. The invention includes a number of proteins isolatable from different C3-degrading strains of S. pneumoniae. 
In one aspect of the invention, the invention relates to an isolated protein comprising at least an 80% sequence identity of SEQ ID NO:2 and capable of degrading human complement protein C3. In a preferred embodiment, the protein is isolated from S. pneumoniae or alternatively the protein is a recombinant protein. Preferably the protein binds human complement protein C3. In a preferred embodiment, the protein has a molecular weight as determined on a 10% polyacrylamide gel of between about 24 kDa to about 34 kDa. A preferred protein of this invention is an isolated protein including SEQ ID NO:2.
The invention also relates to peptides from the C3-degarding proteinase of this invention and preferably peptides of at least 15 sequential amino acids from an isolated protein comprising at least an 80% sequence identity of SEQ ID NO:2 and capable of degrading human complement protein C3 and more preferably peptides of at least 15 sequential amino acids from SEQ ID NO:2.
The protein of claim 9, wherein the protein is a recombinant protein. In another aspect of this invention, the invention relates to a peptide of at least 15 sequential amino acids from SEQ ID NO:2.
The protein of this invention can comprise SEQ ID NO:2, and preferably has a molecular weight as determined on a 10% polyacrylamide gel of between about 24 kDa to about 34 kDa. Also preferably the protein degrades human complement protein C3. Preferred protein or polypeptides of this invention include a protein comprising amino acids 1-50 of SEQ ID NO:2 and a nucleic acid fragment comprising nucleic acids 1246 to 1863 of FIG. 1A.
In another aspect of the invention the invention relates to a protein that degrades human complement protein C3 and wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:1 under hybridization conditions of 6xc3x97SSC, 5xc3x97Denhardt, 0.5% SDS, and 100 xcexcg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65xc2x0 C. and washed in 2xc3x97SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at, 65xc2x0 C. for about 15 minutes followed by at least one wash in 0.2xc3x97SSC, 0.1% SDS at room temperature for at least 3-5 minutes.
The invention also relates to an immune-system stimulating composition comprising an effective amount of an immune system-stimulating peptide or polypeptide comprising at least 15 amino acids from a protein comprising at least an 80% sequence identity with SEQ ID NO:2 and capable of degrading human complement protein C3.
Preferably the protein is isolatable from S. pneumoniae. In one embodiment, the immune system stimulating composition further comprises at least one other immune stimulating peptide, polypeptide or protein from S. pneumoniae. 
The invention further relates to an antibody capable of specifically binding to a protein comprising at least a 80% sequence identity with SEQ ID NO:2 and capable of degrading human complement protein C3. In one embodiment, the antibody is a monoclonal antibody an din an other embodiment, the antibody is a polyclonal antibody. In another embodiment the antibody is an antibody fragment. The antibody or antibody fragments can be obtained from a mouse, a rat, human or a rabbit.
The invention also relates to a nucleic acid fragment capable of hybridizing to SEQ ID NO:1 under hybridization conditions of 6xc3x97SSC, 5xc3x97Denhardt, 0.5% SDS, and 100 xcexcg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65xc2x0 C. and washed in 2xc3x97SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at, 65xc2x0 C. for about 15 minutes followed by at least one wash in 0.2xc3x97SSC, 0.1% SDS at room temperature for at least 3-5 minutes. In one embodiment the nucleic acid fragment is isolated from an S. pneumoniae genome and in another embodiment, the nucleic acid fragment encodes at least a portion of a protein. In one embodiment, the protein degrades human complement C3 and in another embodiment, the nucleic acid fragment encodes a protein that does not degrade human complement C3.
In another embodiment, the nucleic acid fragment is in a nucleic acid vector and the vector can be an expression vector capable of producing at least a portion of a protein. Cells containing the nucleic acid fragment are also contemplated in this invention. In one embodiment, the cell is a bacterium or a eukaryotic cell.
The invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence gctcccagtatgcgtactcgtaaggtagagggaagaaaaaaactagctag. SEQ ID NO:9.
In another aspect of this invention, the invention relates to a method for producing an immune response to S. pneumoniae in an animal including the steps of: administering a composition comprising a therapeutically effective amount of at least a portion of a protein to an animal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:1 under hybridization conditions of 6xc3x97SSC, 5xc3x97Denhardt, 0.5% SDS, and 100 xcexcg/ml fragmented and denatured salmon sperm DNA, hybridized overnight at 65xc2x0 C. and washed in 2xc3x97SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at 65xc2x0 C. for about 15 minutes followed by at least one wash in 0.2xc3x97SSC, 0.1% SDS at room temperature for at least 3-5 minutes; and obtaining an immune response to the protein. In one embodiment the immune response is a B cell response and in another embodiment, the immune response is a T cell response. In a preferred embodiment, the composition is a vaccine composition. Preferably the at least a portion of the protein is at least 15 amino acids in length and also preferably the composition further comprises at least one other protein from S. pneumoniae. In one embodiment, the protein comprises at least 15 amino acids of SEQ ID NO:2.
In a further embodiment, the invention relates to a bacteria comprising an insertional mutation, wherein the insertion mutation is in a gene encoding a protein capable of degrading human complement C3. In one embodiment, the bacteria comprises an insertional duplication mutation.
The invention further relates to an isolated protein of about 24 kDa to about 34 kDa from Streptococcus pneumoniae that is capable of binding to and degrading human complement C3 and to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising the step of: contacting a Streptococcus pneumonia bacterium with antibody capable of binding to a protein with at least 80% amino acid sequence identity to SEQ ID NO:2. The invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO:1 and to an RNA fragment transcribed by a double-stranded DNA sequence comprising SEQ ID NO:1.